ABSTRACT Human herpesvirus 8 (HHV-8) is a causal agent for Kaposi?s sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman?s disease that are often associated with acquired immunodeficiency syndrome. HHV-8 lytic replication, in addition to latency, is important for maintaining viral load within the host as well as for related pathogenesis. Recent evidence shows that HHV-8-encoded viral interferon regulatory factor 1 (vIRF-1) plays an important role in promoting virus replication by suppressing apoptosis and innate immunity elicited by infection of host cells. We recently found, for the first time, that vIRF-1 localizes to mitochondria via a novel mechanism upon virus replication and mitochondria-localized vIRF-1 is essential for successful virus replication. However, the functional significance of mitochondria-localized vIRF-1 to HHV-8 biology and pathogenesis remains to be determined. Our preliminary results showed that mitochondrial content is reduced in lytic vIRF-1-positive PEL cells but restored by pharmacological inhibition of autophagy and mitophagy. Moreover, using recombinant HHV- 8 encoding vIRF-1 with impaired mitochondria targeting and a vIRF-1-derived peptide corresponding to the mitochondrial targeting region, we demonstrated that mitochondria targeting of vIRF-1 plays an important role in regulation of mitochondria content and apoptosis during HHV-8 replication. Our further studies revealed that vIRF- 1 interacts with autophagy-related proteins (GEC1 and EF-Tu) and is subject to posttranslational modifications (caspase-5-mediated cleavage and K63-linked polyubiquitination) upon HHV-8 replication and mitochondrial damage. Moreover, inhibition of mitophagy by Mdivi-1 enhances MAVS-mediated apoptosis induced by virus replication and inhibits HHV-8 productive replication. Based on these findings, we hypothesize that vIRF-1 can sense and respond to mitochondria damage induced by HHV-8 replication and promote removal of dysfunctional mitochondria by activating mitophagy, resulting in inhibition of mitochondria-mediated antiviral responses and promotion of HHV-8 replication. Therefore, an approach to interfere with vIRF-1-activated mitophagy could lead to the development of novel antiviral agents. To further define the mechanisms and functional significance of vIRF-1- activated mitophagy in HHV-8 replication, we propose to: 1) delineate the molecular mechanism(s) of vIRF-1- mediated mitophagy activated by HHV-8 replication, 2) determine the significance to mitophagy regulation of novel posttranslational modifications of mitochondria-localized vIRF-1, and 3) determine the functional significance of vIRF-1-activated mitophagy in MAVS regulation and HHV-8 biology. Successful completion of the studies in this application will delineate the molecular interactions and processes involved in vIRF-1-acitvated mitophagy and provide evidence for the antiviral and therapeutic potentials of mitophagy inhibition. Overall, this proposal will identify a novel paradigm of virus-host interaction via mitochondria and could potentially provide a basis for future development of antiviral agents targeting mitophagy-associated interactions of vIRF-1.